Meningococcal And Pneumococcal Conjugate Vaccine And Method Of Using Same

ABSTRACT

This disclosure relates to vaccine formulations comprising an immunogenic composition for inducing antibodies to both  S. pneumoniae  and  N. meningitides  in a subject. In a preferred aspect, the immunogenic composition comprises covalently conjugated recombinant PsaA (“rPsaA”) from  S. pneumoniae  and capsular polysaccharide from  N. meningitidis  serogroup C. This disclosure further relates to methods for producing the immunogenic composition as well as methods for their use.

RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.13/183,303, filed Jul.14, 2011, which is a continuation of U.S.application Ser. No. 12/425,232, filed Apr. 16, 2009, which areincorporated herein by reference in their entireties.

FIELD OF THE INVENTION

This disclosure relates to vaccine formulations that include animmunogenic composition for inducing antibodies to the S. pneumoniaePsaA protein and N. meningitidis capsular polysaccharide. Thisdisclosure further relates to methods for producing the immunogeniccomposition as well as methods for their use.

BACKGROUND OF THE INVENTION

Status of current pneumococcal vaccines. S. pneumoniae is agram-positive encapsulated diplococcus. Capsule, a layer ofpolysaccharide (PS) surrounding the bacterial cell, is a major virulencefactor of S. pneumoniae. Based on the differences in structure andimmunological response to capsular polysaccharide, S. pneumoniae can bedivided into more than 90 different serotypes. Capsular polysaccharidesare the base for the currently used vaccines. The FDA has approved twotypes of pneumococcal vaccines for use in humans: a 23-valent PS vaccineand a 7-valent PS/protein conjugate vaccine. The former is comprised ofcapsular polysaccharide purified from 23 different serotypes of S.pneumoniae, which account for almost 89 percent of disease cases.PNEUMOVAX® (Merck) is an example of this group of vaccines. However, PSelicits type-specific antibodies. Antibodies raised for one serotype donot provide protection against infection of other serotypes. Theefficacy of the 23-valent vaccine is limited. Furthermore, PS is aT-cell independent antigen which induces short-term immunity withoutimmune memory and is not effective in children younger than two years ofage (Greenwood B M et al., Trans R Soc Trop Med Hyg, 1980, 74:756-760).It is only recommended for high risk groups, such as the elderly andpersons with underlying disease. A recently approved pneumococcalvaccine is a mixture of conjugates of 7 different individually preparedcapsular polysaccharides covalently linked with carrier protein CRM197,which is a non-toxic and immunologically cross-reactive mutant ofdiphtheria toxin (Uchida et al, J. Biol. Chem. 248:3838-3844, 1973) anda component of the pediatric DPT (Diphtheria-Tetanus-Pertussis toxin)vaccine. Upon conjugation to a carrier protein, the otherwise T-cellindependent PS becomes a T-cell dependent antigen by obtaining theimmunological property of the protein. (Schneerson R et al., J Exp Med1980, 152:361-376). The conjugate induces long-lasting immunity withimmune memory and is effective in young infants. The 7 serotypes wereselected for their prevalence in pediatric diseases. A conjugate vaccineof 7 pneumococcal capsular PS (PCV7) with CRM197 (Wyeth) is the onlyvaccine of this family that is commercially available. It is onlyprescribed for use in the prevention of pediatric invasive pneumococcaldisease because of its high cost and limited supply. The drawback ofthese two families of vaccines is that they only provide protectionagainst infection by the specific serotypes of S. pneumoniae that areincluded in the respective vaccine formulations.

Status of current meningococcal vaccine. N. meningitidis is agram-negative, encapsulated diplococcus. At least 13 differentserogroups have been identified based on the structure of capsular PS,but serogroups A, B, C, Y, and W-135 account for almost all cases ofdisease. Serogroup B organisms account for 46 percent of all cases,serogroup C for 45 percent of all cases, and serogroups W-135 and Y andstrains that could not be serogrouped account for most of the remainingcases. Like S. pneumoniae, the major ingredient for meningococcalvaccines is capsular PS. Its vaccines can be divided into two families:the capsular PS vaccine and PS-protein conjugate vaccines. Threeversions of PS vaccines are commercially available.

Quadrivalent PS vaccine (GlaxoSmithKline and Sanofi-Pasteur) is composedof capsular PS purified from serogroups A, C, Y, and W-135. It isexpensive and not affordable for developing countries. Bivalent PSvaccine (GlaxoSmithKline and Sanofi-Pasteur) is composed of capsular PSpurified from serogroups A and C. Trivalent PS vaccine (GlaxoSmithKline)is composed of capsular PS purified from serogroups A, C, and W-135.This vaccine has been used in the epidemics in the “Meningitis Belt”countries in Africa. Like pneumococcal vaccine, PS vaccine is notefficacious in children younger than two years of age. Such deficiencycan be overcome by PS-protein conjugates.

Two types of meningococcal vaccine conjugates are commercially availableor being developed. MENACTRA® (Sanofi-Pasteur) is the first quadrivalentconjugate meningococcal vaccine. It is a mixture of meningococcalpolysaccharides (groups A, C, Y, and W135) conjugated with diphtheriatoxoid. A monovalent meningococcal conjugate vaccine currently underdevelopment is a conjugate of serogroup C polysaccharide-diphtheriatoxoid (Chiron and Wyeth), serogroup C PS-tetanus toxoid (Chiron,Baxter), and serogroup A PS-tetanus toxoid (PATH-SII). Preliminaryresults of clinical trials indicate these vaccines are efficacious.

With the burden of S. pneumoniae and N. meningitidis infection on thepublic health system at a global scale, it is desirable to have a singlevaccine that is effective to prevent disease resulting from theinfection of both pathogens.

SUMMARY

This disclosure provides an immunogenic composition for inducing animmune response to two different microorganisms, S. pneumoniae and N.meningitidis. This disclosure further provides an inoculum and/orvaccine comprising the immunogenic composition dispersed and/ordissolved in a pharmaceutically acceptable diluent. The vaccine includesat least one N. meningitidis capsular polysaccharide conjugated to apneumococcal protein. In a preferred aspect, the immunogenic compositioncomprises recombinant PsaA (“rPsaA”) from S. pneumoniae and capsularpolysaccharide from N. meningitidis serogroup C. Pneumococcal proteinacts as an antigen as well as a carrier protein for N. meningitidiscapsular polysaccharide in the vaccine. Thus, the vaccine is effectivefor providing dual protection against infection by both S. pneumoniaeand N. meningitidis.

Several pneumococcal proteins are universally found in all testedserotypes of S. pneumoniae, such as pneumococcal surface antigen A(PsaA), pneumococcal surface protein A (PspA), pneumococcal surfaceprotein C (PspC), pneumolysin, and histidine-triad proteins. Studieshave shown that these proteins are capable of eliciting protectiveantibodies in laboratory animals. In particular, PsaA has been found byimmunological and PCR methods in all S. pneumoniae tested including 23vaccine serotypes as well as clinical isolates from various countries.PsaA has a length of 309 amino acid residues. In an important aspect,the rPsaA used in the immunogenic composition described herein includesat least the amino acid residues at positions 21 to 319 of SEQ ID NO:1.

The capsular polysaccharide (about 300,000 Da) of N. meningitidisserogroup C comprises about 850 repeating units of sialic acid withα(2→9) glycosidic linkage and about 80 percent O-acetylation at C7 orC8. The capsular polysaccharide of N. meningitidis serogroup C and PsaAare provided in conjugated form. In a preferred aspect, the capsularpolysaccharide and PsaA are conjugated by covalent linkage.

In another aspect, a method is provided for generating an immuneresponse in a subject against pneumococcal surface antigen A (PsaA) andcapsular polysaccharide from N. meningitidis serogroup C. The methodcomprises administering to a subject an effective amount for inducingproduction of antibodies specific to rPsaA and capsular polysaccharidefrom N. meningitidis serogroup C. Administering to a subject acombination of rPsaA and capsular polysaccharide from N. meningitidisserogroup C in covalently linked form is effective for generating animmune response in the subject. In an important aspect, immunogenicityof the conjugated pneumococcal surface antigen A (PsaA) and capsularpolysaccharide is significantly increased as compared to the immuneresponse observed when the antigens are administered individually. Inthis aspect, more than a 40-fold increase in immunogenicity is seen forconjugated PsaA as compared to non-conjugated PsaA, and more than a170-fold increase in immunogenicity is seen for conjugated capsularpolysaccharide as compared to non-conjugated capsular polysaccharide.

The immunogenic composition may be administered to a subject by a numberof different routes, including intramuscular administration, intranasaladministration, oral administration, sub-cutaneous administration,transdermal administration, and transmucosal administration.

Immunogenic compositions described herein are prepared by a methodcomprising preparing recombinant PsaA (“rPsaA”) and conjugating rPsaAwith capsular polysaccharide from N. meningitidis serogroup C. rPsaA canbe prepared using well-known recombinant techniques. Capsularpolysaccharide can be isolated from natural sources or synthesized usinga number of techniques which are well known in the art.

The immunogenic compositions described herein advantageously providedual protection against S. pneumoniae and N. meningitidis infection. Theimmunogenic composition described herein also utilizes PsaA as a proteincarrier for polysaccharide.

Advantageously, the conjugated immunogenic composition provided hereincan reduce the costs of preparing and administering the vaccine. This isa particularly important benefit to developing and underdevelopedcountries because the vaccine will reduce the economic and medicalburden to the countries which have high rates of pneumococcal andmeningococcal disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides the nucleotide sequence (SEQ ID NO. 2) of a cloned psaAfragment, including restriction endonuclease sites at the 5′ and 3′ endsproduced according to the Example.

FIG. 2 provides the deduced amino acid sequence (SEQ ID NO. 1) ofrecombinant PsaA protein produced according to the Example.

FIG. 3 shows a photograph of a SDS-polyacrylamide gel electrophoresisand Western blot analysis of rPsaA according to the Example.

FIG. 4 is a chromatogram demonstrating that the protein signal shiftedfrom a low molecular weight position to a high molecular weight for theconjugate produced according to the Example.

FIG. 5A shows a photograph of an Immuno-dot blot according to theExample. FIG. 5B shows a photograph of a Western blot according to theExample.

DETAILED DESCRIPTION

This disclosure provides an immunogenic composition comprising capsularpolysaccharide from N. meningitidis and a protein from S. pneumoniae(referred to as “Pn-Mn” vaccine). In a preferred aspect, the S.pneumoniae protein is recombinant pneumococcal surface antigen A(“rPsaA”) and the N. meningitidis capsular polysaccharide is serogroup Ccapsular polysaccharide. PsaA is universally found in all testedserotypes of S. pneumoniae. The immunogenic composition is useful forinducing production of antibodies for diagnostic and therapeuticpurposes. This disclosure further provides an inoculum and vaccinecomprising the immunogenic composition dispersed or dissolved in apharmaceutically acceptable diluent. It is particularly preferred thatthe rPsaA from S. pneumoniae is covalently conjugated to capsularpolysaccharide from N. meningitidis serogroup C.

The term “antibody” refers to a molecule that is a member of a family ofglycosylated proteins called immunoglobulins, which can specificallybind to an antigen. The word “antigen” refers to an entity that is boundby an antibody. “Immunogen” or “immunogenic composition” refers to theentity that induces antibody production or binds to the receptor.

The words “protein” and “polypeptide” are used interchangeablythroughout the specification and designate a series of amino acidresidues connected by peptide bonds.

Capsular polysaccharide from N. meningitidis Serogroup C

Polysaccharide is a T cell-independent (T-I) antigen inducing short-termimmunity with little immune memory and is not effective in infantsyounger than 2 years old. When covalently linked to a carrier protein,the resulting PS component in a conjugate vaccine becomes a Tcell-dependent (T-D) antigen inducing long-term immunity with immunememory even in infants and young children.

The capsular polysaccharide of N. meningitidis serogroup C comprisesrepeating units of sialic acid with a (2→9) glycosidic linkage and about80 percent O-acetylation at C7 or C8. The size of the N. meningitidisgroup C polysaccharide is about 590 to about 1,030 sialic acid repeatingunits assuming the molecular weight of a sialic acid repeating unit is340 Daltons. The size of the N. meningitidis serogroup C capsularpolysaccharide particularly useful in the invention is about 200 toabout 350 kDa, preferably about 250 to about 300 kDa, although othersizes may be used, if desired, provided that the selected size of thepolysaccharide is effective to induce production of antibodies in asubject after conjugation to a carrier protein.

The capsular polysaccharide can be isolated from natural sources using anumber of techniques which are well known in the art. For example, N.meningitidis group C strain can be grown in a defined medium for 18hours and inactivated with 0.5 percent formaldehyde. Aftercentrifugation to precipitate the cells, the polysaccharide in theremoved supernatant can be precipitated by 0.1 percent cetavlon. Theinsoluble cetavlon complex is then dissolved in 0.9 M calcium chlorideand the crude polysaccharide is precipitated with 5 volume ethanol. Theprecipitate is further dissolved in phosphate buffer. After phenolextraction and ribonuclease treatment, the sample is dialyzed againstwater and concentrated (Bundle et al, J. Biol. Chem. 249:4797-4801,1974, which is incorporated herein by reference.)

In another aspect, the capsular polysaccharide derived from N.meningitidis serogroup C may be substituted with capsular polysaccharidederived from N. meningitidis serogroups A, B, D, X, Y, Z, 29E, W-135, ora combination thereof, in the Pn-Mn conjugates described herein. N.meningitidis serogroups A, B, C, D, X, Y, Z, 29E, and W-135 account foralmost all cases of disease. Such conjugates can be administered to asubject capable of inducing an immune response to an antigen in order toprovide protection against infection of these serogroups. Meningococcalserogroup A polysaccharide (about 300 kDa) is composed of N-acetylmannosamine 6-phosphate repeating units with α (1→phosphate) glycosidiclinkage and about 70-90 percent O-acetylation at C3. Meningococcalserogroup W135 polysaccharide (˜300,000 Daltons) is composed of (2→6)α-D-galactose (1→4) α-D-sialic acid repeating units with about 70percent O-acetylation at C7 or C9 of the sialic acid residue.Meningococcal serogroup Y polysaccharide (about 300 kDa) is composed of(2→6) α-D-galactose (1→4) α-D-sialic acid repeating units with about 70percent O-acetylation at C7 or C9 of the sialic acid residue. The sizeof the N. meningitidis capsular polysaccharide particularly useful inthe invention is about 200 to about 350 kDa, preferably about 250 toabout 300 kDa, although other sizes may be used, if desired, providedthat the selected size of the polysaccharide is effective to induceproduction of antibodies in a subject after conjugation to a carrierprotein. The activation conditions for these polysaccharides may bedifferent from that for group C polysaccharide due to differences intheir structures.

Pneumococcal Protein

PsaA has a length of 309 amino acid residues. It is preferred that therPsaA used in the immunogenic composition includes at least the residuesat positions 21 to 319 of SEQ ID NO:1.

Recombinant PsaA from S. pneumoniae can be prepared using conventionalrecombinant techniques. Recombinant methodologies required to produce aDNA encoding a desired protein are well known and are routine to thoseof ordinary skill in the art. The nucleic acid sequences used topractice this invention, whether cDNA, genomic DNA, vectors, and thelike, may be isolated from a variety of sources, genetically engineered,amplified, and/or expressed recombinantly. The nucleotide sequence forpsaA is provided at nucleotide positions 6 to 867 in SEQ ID NO:2. Thecoding sequence of the desired protein can be cloned into a vector.

Any recombinant expression system can be used, including bacterial,mammalian, yeast, insect, or plant cell expression systems.Alternatively, these nucleic acids can be synthesized in vitro bywell-known chemical synthesis techniques. Double stranded DNA fragmentsmay then be obtained either by synthesizing the complementary strand andannealing the strands together under appropriate conditions, or byadding the complementary strand using DNA polymerase with an appropriateprimer sequence.

Nucleic acid amplification methods are well known in the art.Oligonucleotide primers can be used to amplify nucleic acids to generatepsaA coding sequence used to prepare recombinant PsaA. The codingsequence can be cloned into an expression cassette, such as plasmids,recombinant viruses which can infect or transfect cells in vitro, exvivo, and/or in vivo, and other vectors which can be used to express thePsaA polypeptide in vitro or in vivo. Selection markers can beincorporated to confer a selectable phenotype on transformed cells, suchas antibiotic resistance. The expressed rPsaA can be recovered andpurified using conventional techniques.

In another aspect and in addition to PsaA, other pneumococcal proteinscan be used as a component of the Pn-Mn conjugate vaccine providedherein. Other S. pneumoniae proteins that may be used includepneumolysin, pneumococcal surface protein A (PspA), pneumococcal surfaceprotein C (PspC or CbpA), pneumococcal histidine triad proteins orsimilar proteins with different nomenclatures such as PhtA or BVH11-3,PhtB or PhpA or BVH-11, PhtE or BVH-3), PhtD or BVH-11-2, and,pneumococcal choline binding protein A (PcpA), non-heme iron-containingferritin or pneumococcal protective proteins (PppA, Dpr), neuraminidaseA (NanA), neuraminidase B (NanB), iron transport proteins oriron-compound-binding protein PiuA and PiaA, N-acetylmuramoyl-L-alanineamidase or autolysin (LytA), endo-β-acetylglucosaminidase (LytB),1,4-β-N-acetylmuranminidase (LytC), caseinolytic protease or serineproteases (ClpP), and adherence and virulence protein A (PavA).

Conjugate Preparation

Polysaccharides contain hydroxyl groups, and occasionally carboxyl andamino groups, and proteins contain amino and carboxyl groups. Bothpolysaccharides and proteins are not active for chemical reaction witheach other in their natural form. Proper pretreatment or activation ofone or both of the polysaccharide and protein is required to convert theotherwise non-reactive molecules to a reactive form in order to producethe polysaccharide-protein conjugate. Many methods are known in the artfor conjugating a protein to a polysaccharide. Polysaccharide can beactivated by cyanogen bromide to provide cyanate groups which react withhydrazide-activated protein (Schneerson et al., J. Exp. Med. 1980;152:361-3760). Polysaccharide can be activated by cyanogen bromide toprovide cyanate groups, which further reacts with di-hydrazide, and thenconjugates to protein in the presence of EDC (Chu et al., Infect. Immun1983; 40:245-256). Polysaccharide can be partially hydrolyzed and addedwith an amino group at the reducing terminus. After a bifunctionallinker is added to the amino group, the activated polysaccharide isconjugated to the carrier protein (Costantino et al., Vaccine 1992;10:691-8). Polysaccharide can be activated with1-cyano-4-dimethylaminopyridinium tetrafluoroborate to provide cyanategroups which react with a carrier protein (Lees A, Nelson B L, Mond JJ.Vaccine 1996; 14:190-198).

In a preferred aspect, rPsaA is dialyzed before use, such as against 30mM NaCl at about 4° C. for about 18 to about 24 hours. The dialyzedrPsaA is then treated to activate the protein, such as with 0.1 M MES(pH 6.5), 0.5 M hydrazine (pH 7.0), and 20 mM 1-[3-dimethylaminopropyl)-3-ethyl carbodiimide-HCl (“EDC” from Sigma-Aldrich) in saline,and incubated for 4 hours. The treated rPsaA is then neutralized, suchas with 1 M NaOH, before dialyzing the protein, such as dialyzingagainst buffer containing 3 mM Na₂CO₃ and 30 mM NaCl at 4° C. Thedialyzed activated rPsaA can be used immediately or stored at 4° C.

In a preferred aspect, the capsular polysaccharide is treated with 6 mMsodium periodate and incubated for 4 hours at room temperature toactivate the capsular polysaccharide. The activated capsularpolysaccharide is then dialyzed against deionized water, such as forabout 18 to about 24 hours at 4° C. The dialyzed activated capsularpolysaccharide can be used immediately or stored at 4° C.

Activated rPsaA is lyophilized, redissolved in water. Dialyzed activatedcapsular polysaccharide (is lyophilized, redissolved in 0.2 M HEPES, pH7.5, 30 mM EDTA. The protein solution is added to the polysaccharidesolution and incubated overnight. NaBH₄ is added to a finalconcentration of 50 mM and incubated for about 4 to about 6 hours toreduce the C═N double bonds in the polysaccharide-protein conjugate toC-N single bonds, and to reduce the unreacted aldehyde to alcohol. Theconjugate is dialyzed against 150 mM NaCl, 10 mM HEPES (pH 7), 1 mM EDTAat 4° C. The dialyzed conjugate can then be evaluated, such as by HPLC,for shift of protein signal (280 nm) from 19 minute position to 18minute upon conjugation.

Method of Using Conjugate

The rPsaA/capsular polysaccharide conjugate provided herein can beadministered to a subject capable of inducing an immune response to anantigen. The rPsaA/capsular polysaccharide conjugate is administered tothe subject in an effective amount for inducing an antibody response. An“effective amount” is an amount of rPsaA/capsular polysaccharideconjugate which assists a subject in producing both anti-rPsaA andanti-capsular polysaccharide antibodies. Such antibodies may preventinfection by S. pneumoniae and N. meningitidis serotype C.

One of ordinary skill in the art can determine whether an amount of therPsaA/capsular polysaccharide conjugate is effective to induce immunityin a subject using routine methods known in the art. For example, theability of an antigen to produce antibody in a subject can be determinedby screening for antibodies using separate coating antigens rPsaA andcapsular polysaccharide in the respective ELISA assays.

In one aspect, a vaccine formulation is provided for N. meningitidisserogroup C and S. pneumoniae. The vaccine formulation is effective forgenerating an immune response in a subject to both N. meningitidisserogroup C and S. pneumonia. The vaccine formulation comprises rPsaAfrom S. pneumoniae and capsular polysaccharide from N. meningitidisserogroup C. The conjugated immunogenic composition can be provided withone or more additional components, such as a pharmaceutically acceptablediluents, carriers, adjuvants, and/or buffers. For example, theconjugate can be dispersed or dissolved in a diluent.

The immunogenic composition may be prepared as a solution, suspension,tablet, pill, capsule, sustained release formulation, powder, or thelike. The antigens and immunogenic composition may be mixed withphysiologically acceptable carriers which are compatible therewith.These may include water, saline, dextrose, glycerol, ethanol,combinations thereof, and the like. The vaccine may further containauxiliary substances, such as wetting or emulsifying agents or pHbuffering agents, to further enhance the effectiveness. Administrationof the conjugate in a vaccine formulation can include delivery byvarious routes, such as, for example, oral, intravenous, intramuscular,nasal, subcutaneous, and intraperitoneal administration.

The immunogenic composition is administered in a manner compatible withthe dosage formulation, and in such amount as to be therapeuticallyeffective, protective, and immunogenic. The quantity to be administereddepends on the subject to the immunized, including, for example, thecapacity of the subject's immune system to synthesize antibodies and, ifneeded, to produce a cell-mediated immune response. Precise amounts ofantigen and immunogenic composition to be administered depend on thejudgment of the practitioner. However, suitable dosage ranges arereadily determinable by those skilled in the art and may be of the orderof micrograms to milligrams. Suitable regimes for initial administrationand booster doses are also variable but may include an initialadministration followed by subsequent administrations. The dosage of thevaccine may also depend on the route of administration and will varyaccording to the size of the subject.

In an important aspect, the rPsaA/capsular polysaccharide Pn-Mnconjugate provided herein may be used to prevent infection of both S.pneumoniae and N. meningitidis serotype C, which are the leading causesof otitis media and meningitis in young children. Furthermore, therPsaA/capsular polysaccharide conjugate provided herein also could beused in the prevention of other pneumococcal and meningococcal diseases,such as bacteremia, pneumoniae and meningitis in the population of otherage groups.

The examples that follow are intended to illustrate the invention andnot to limit it. All percentages used herein are by weight unlessotherwise indicated. All patents, patent applications, and literaturereferences cited herein are hereby incorporated by reference in theirentirety.

EXAMPLE

A better understanding of the vaccine provided herein and its manyadvantages is provided with the following example.

A. Preparation of Purified rPsaA

psaA gene cloning and expression. To prepare recombinant pneumococcalPsaA (rPsaA) protein, the coding sequence of pneumococcal psaA genes inE. coli was cloned in the expression vector pET22b(+) (Novagen, Madison,Wis.). Sequence analysis revealed that the coding sequence of psaA doesnot include BamHI and HindIII restriction sites. For the purpose ofcloning, expression, and purification of rPsaA protein, a pair ofprimers for PCR amplification were designed so that: 1) the PCR productwould have a BamHI and HindIII site at the 5′ and 3′ ends, respectively;2) the reading frame of cloned psaA would be in-frame with that of thevector; and 3) the produced rPsaA protein would have a His-tag at itsC-terminal. The forward and reverse primers(5′-GGGATCCTAGCGGAAAAAAAGATACA-3′ (SEQ ID NO. 3),5′-GCAAGCTTTGCCAATCCTTCAGCAATC-3′ (SEQ ID NO. 4), respectively, wereintended to amplify a 868-bp fragment starting from nucleotide no. 42 tono. 921 of the psaA coding sequence. The underlined nucleotides indicatethe positions of BamHI and HindIII sites in these primers. The codedrPsaA protein would have 331 amino residues and a predicted molecularmass of 36,940 daltons. The nucleotide sequence of the cloned fragmentis shown in FIG. 1 and the predicted amino acid sequence for rPsaA inFIG. 2.

A typical PCR mixture contained 5 μmole primers, 20 ng S. pneumoniaeserotype 4 chromosomal DNA and PCR Supermix (Life Technologies,Rockville, MD). The conditions for PCR were as follows: DNA denaturationat 95° C. for 40 seconds, primer annealing at 42° C. for 1 min, and DNAsynthesis at 72° C. for 1.5 min. After 30 cycles of synthesis, thereaction was terminated with an extension at 72° C. for 5 min. The PCRproducts were purified with the GeneClean kit (Qbiogen, Carlsbad,Calif.), cloned into pGEM-T easy vector (Promega, Madison, Wis.) andtransformed into E. coli DH5α. The insert was isolated from theresultant plasmid after a double digestion with restriction enzymesBamHI and HindIII, cloned into the compatible site of pET22b(+) togenerate plasmid pST648, and transformed into E. coli BL21(DE3). Toconfirm that psaA gene on pST648 was cloned as planned, the restrictionmap of the cloned PCR product was determined. The results wereconsistent with those of published psaA gene. The proper cloning of theBamHI-HindIII restriction fragment into pET22b(+) was further confirmedby the induction of recombinant protein and by the presence of His-tagat the carboxyl end.

To induce the synthesis of recombinant protein,isopropyl-13-D-thiogalactoside (IPTG, 0.1 mM) was added to the log-phaseculture (A600 nm^(=0.6)) of E. coli BL21(DE3) harboring pST648 andgrowth continued for another 2 hours. Cells were harvested, washed,suspended in one-tenth volume of 50 mM Tris-HCl, pH 7.9 containing 200mM NaCl (TN buffer) at 4° C., and disrupted by sonication. After theremoval of unbroken cells by centrifugation, the supernatant was subjectto SDS-PAGE analysis. To confirm that recombinant protein had a His-tag,the proteins on the SDS-gel were analyzed by western blotting againstmouse monoclonal anti-poly-histidine antibody (Sigma-Aldrich, St. Louis,Mo.). The proteins on the gel were transferred onto nitrocellulose paperand the paper was washed with blotto (20 mM Tris, 0.2M NaCl, 1.5 percentnonfat milk), incubated with monoclonal anti-poly-histidine antibody inblotto (1:200 dilution) for 2 hours, washed with blotto three times,incubated with alkaline phosphatase-conjugated goat anti-mouse antibodyin blotto (1:5000 dilution), and washed with blotto and AP buffer (0.1 MNaCl, 0.1 M Tris-Cl, pH 9.5). The antibody-antigen interaction wasvisualized by incubating with 0.1 percent naphthol and 1 percent fastblue (Sigma-Aldrich). The results are shown in FIG. 3. The resultsindicate that the overproduced protein was indeed rPsaA and the crudecell lysate of E. coli BL2I(DE3)(pST648) could be used as the source ofrPsaA in protein purification.

Purification of rPsaA protein. To purify rPsaA protein, crude celllysate was loaded on a HIS-BIND® Column (Novagen, Madison, Wis.). Theresin was washed with binding buffer (TN buffer containing 50 mMimidazole) and washing buffer (TN buffer containing 200 mM imidazole) toremove excess and nonspecifically bound proteins. The bound protein waseluted with elution buffer (TN buffer containing 1 M imidazole) andanalyzed by SDS-polyacrylamide gel electrophoresis and Western blottingagainst monoclonal anti-poly-histidine antibody as described above.Fractions containing protein that reacts with anti-poly-histidinemonoclonal antibody were collected as purified rPsaA protein (FIG. 3).

B. Preparation of rPsaA-MCPS Conjugate

Activation of rPsaA. rPsaA was dialyzed against 30 mM NaCl at 4° C.overnight before use. The dialyzed rPsaA was mixed with 1 M MES, pH 6.5,5 M hydrazine, pH 7.0, 1 M EDC (Sigma-Aldrich) in saline at the finalconcentration of 0.1 M, 0.5 M, and 20 mM, respectively. After incubationat room temperature for 4 hours, 1 M NaOH (0.05 mL) was added toneutralize the reaction before dialysis against buffer containing 3 mMNa₂CO₃ and 30 mM NaCl at 4° C. The protein solution was stored at 4° C.

MCPS activation. N. meningitidis type C capsular PS (MCPS, 10 mg/ML) wasmixed with sodium periodate at a final concentration of 6 mM. Afterincubation at room temperature for 4 hours, the reaction mixture wasdialyzed against deionized water overnight and stored at 4° C.

Conjugation of PsaA-MCPS. Aliquot activated rPsaA (0.25 mg) waslyophilized and re-dissolved in 25 μl water. Aliquot activated MCPS(0.25 mg) was lyophilized and redissolved in 25 μl of 0.2 M HEPES, pH7.5 containing 30 mM EDTA. These two solutions were combined. Afterincubation overnight at room temperature, 5 μl of 1 M NaBH₄ was addedand incubation continued for another 6 hours. After dialysis against 150mM NaCl, 10 mM HEPES, pH 7, 1 mM EDTA at 4° C., the conjugate productwas stored at 4° C. The conjugate of MCPS with rPsaA was evaluated withHPLC analysis using a Waters Ultrahydrogel Linear size-exclusion columnand monitored at the wavelengths of 206 nm and 280 nm. Upon conjugation,the protein signal shifted from low molecular weight position to thehigh molecular weight in the chromatogram, as shown in FIG. 4.

C. Characterization of rPsaA-MCPS Conjugate.

Immunogenicity. Mice (NIH-Swiss) were subcutaneously immunized every twoweeks with rPsaA, MCPS, or PsaA-MCPS conjugate, respectively, at thedose of 1 μg per mouse. Blood was collected from optical vein two weeksafter the third immunization and the titers of antibodies weredetermined by enzyme-linked immunosorbent assay (ELISA). Briefly, wellsof microtiter plate (Dynatec, no.1) was coated with MCPS by adding 100μl of solution comprised of antigen, 0.5 μg/mL rPsaA or 5 μg/mL nativeMCPS plus 5 mg/ml methylated human serum albumin in PBS, pH 7.5 andincubated at room temperature for at least 4 hours. Wells were washedthree times (150 μl/well) with PBS containing 0.05 percent TWEEN® 20 and0.02 percent NaN₃. 100 μL of diluent (5 percent calf serum and 0.02percent NaN₃ in PBS) was added to each well and a two-fold serialdilution of diluted (1:100) antiserum was prepared. The reference serum,which was assigned with 3,200 units/mL IgG against MCPS or rPsaA, wassimilarly treated in the same plate. After incubating overnight at roomtemperature and washing three times, 100 μl of alkalinephosphate-conjugated goat anti-mouse IgG Fc (1:3000 dilution) was addedand incubated at room temperature for 3 hours. Wells were washed threetimes and 100 μl of substrate (p-nitrophenyl phosphate, 1 mg/mL in 1 MTris-HCl, pH 9.8 containing 0.3 mM MgCl₂) was added. The plate wasincubated at room temperature for 20 minutes (it might vary depending onthe color development of sample and reference serum) and the absorbanceswere measured at 405 nm. The respective reference serum for MCPS andrPsaA was prepared in the laboratory and were used as standards todetermine the antibody level of the sample serum. Results are shown inTable I below.

TABLE I Immunogenicity of rPsaA, MCPS, and rPsaA-MCPS conjugate. IgGlevel* Antigen Dose anti-PsaA anti-MCPS rPsaA 3 × 1 μg 107 (9; 1678)   —MCPS 3 × 1 μg — 533 (46; 6176) rPsaA-MCPS 3 × 1 μg 4,418 (2006; 9734)90,506 (50,421; 162,455) *The data that is not in parenthesis representsthe geometric mean of IgG antibody level in 10 antiserum samples. Theanti-rPsaA or anti-MCPS IgG antibody level of each antiserum wasmeasured by ELISA and compared with respective reference serum, assignedwith 3,200 unit/mL IgG antibody. The numbers in parenthesis representsthe confidence interval of one standard deviation.

Both rPsaA and MCPS were immunogenic in mice in the absence of adjuvant.Their immunogenicity increased significantly after they were conjugated.When compared with each individual component, the immunogenicityincreased approximately 41-fold and 170-fold for rPsaA and MCPS,respectively.

Reactivity of anti-rPsaA antibodies. It has been demonstrated thatactive immunization of PsaA is effective to protect laboratory animalsfrom S. pneumoniae infection. To provide protection, anti-PsaA shouldinteract with all S. pneumoniae cells. The cross-reactivity of thegenerated anti-rPsaA antibodies was investigated by immuno-dot blottingand western blotting against clinical isolates of S. pneumoniae,including serotypes 1, 2, 3, 4, 5, 6A, 6B, 7C, 8, 9A, 10A, 10B, 11A,12A, 14, 15A, 15C, 16F, 18A, 18C, 19A, 19F, 20, 24, 22A, 23B, 23F, 23Cand 35. Cells of S. pneumoniae were cultured in 15 mL Todd-Hewitt brothovernight at 37° C. in the presence of 5 percent CO2, harvested bycentrifugation, and suspended in 2 mL of TN buffer. Cells were disruptedby sonication in ice bath at the energy level of 7, 50 percent cycle,for 5 minutes. The supernatant after centrifugation at 10,000×g for 10minutes was collected and used as the source of S. pneumoniae proteins.For immuno-dot blotting, 5 μl cell lysate was spotted on thenitrocellulose paper. For Western blot, randomly selected pneumococcalcell lysates were analyzed by SDS-PAGE and transferred on nitrocellulosepaper. The paper was processed as described above, except anti-rPsaAantibody was used. Results are shown in FIG. 5A and FIG. 5B. Theanti-rPsaA antibody cross-reacted with cells of all serotypes tested andreacted with a single protein that has an apparent molecular weightcomparable to that of PsaA.

Bactericidal activity of anti-MCPS antibody. The biological function ofthe induced MCPS-specific antibodies was determined by bactericidalassay against N. meningitidis serogroup C (strain C11). Briefly,bacteria were cultured overnight on brain heart infusion (BHI) agarplates containing 5 percent normal horse serum (NHS) and transferred tofresh plates and cultured for 5 hours the second day. Bacteria from the5 hour culture were suspended to 65-66 percent transmittance at 530 nmin DPBSG (1×PBS, pH 7.2, 0.5 mM MgCl₂, 0.9 mM CaCl₂, and 0.01 percentgelatin) followed by 1:10,000 dilution with the same buffer to containapproximately 4,000 cfu/mL. In the wells of a microtiter plate, 50 μl2-fold dilutions of test and control sera were prepared with DPBSG andmixed with 25 μl bacterial suspension and 25 μl baby rabbit complement(Pel-Freez, Rogers, Ark.). After incubation at 37° C. for 60 min, 10 μlof the bacterial suspension was withdrawn from each well and spread onthe BHI/NHS plate. The colonies were enumerated after incubationovernight at 37° C. with 5 percent CO₂. The bactericidal titer was thereciprocal of the highest dilution of the sample yielding a 50 percentreduction in CFU as compared to the control well containing complementwithout antiserum. The geometric means of the titer for each mouse groupwas calculated. Results are shown in Table II below.

TABLE II Bactericidal activity of antisera against MCPS, rPsaA-MCPSconjugate. Antigen Bactericidal activity titer* MCPS 109 (63; 190)rPsaA-PCPS 5022 (1123; 22454) *The data that is not in parenthesisrepresents the geometric mean of sera from 10 mice for each antigen. Thenumbers in parenthesis represents the confidence interval of onestandard deviation.

Sera for both MCPS and rPsaA-MCPS conjugates had bactericidal activity,but the titer for the conjugates were significantly higher(approximately 46-fold).

While the invention has been particularly described with specificreference to particular process and product embodiments, it will beappreciated that various alterations, modifications, and adaptations maybe based on the present disclosure, and are intended to be within thespirit and scope of the invention as defined by the following claims.

What is claimed is:
 1. A vaccine comprising capsular polysaccharide fromN. meningitidis conjugated to a pneumococcal protein.
 2. The vaccine ofclaim 1 wherein the N. meningitidis capsular polysaccharide is derivedfrom at least one N. meningitidis serogroup selected from the groupconsisting of serogroups A, B, C, D, X, Y, Z, 29E, W-135, andcombinations thereof.
 3. The vaccine of claim 2 wherein the N.meningitidis capsular polysaccharide is derived from N. meningitidisserogroup C.
 4. The vaccine of claim 1 wherein the pneumococcal proteinis selected from the group consisting of PsaA, pneumolysin, PspA, PspC,CbpA, pneumococcal histidine triad protein selected from the groupconsisting of PhtA, BVH11-3, PhtB, PhpA, BVH-11, PhtE, BVH-3, PhtD andBVH-11-2, PcpA, PppA, Dpr, NanA, NanB, PiuA, PiaA, LytA, LytB, LytC,ClpP, PavA, and combinations thereof.
 5. The vaccine of claim 4 whereinthe pneumococcal protein is PsaA.
 6. A method for generating an immuneresponse against N. meningitidis and S. pneumoniae in an individual, themethod comprising administering to the individual an amount of vaccinecomposition effective for generating an immune response to both N.meningitidis and S. pneumoniae, wherein the vaccine compositioncomprises N. meningitidis capsular polysaccharide conjugated to apneumococcal protein.
 7. The method of claim 6 wherein the vaccine isadministered by a route selected from the group consisting of oralintravenous, intramuscular , nasal, subcutaneous, intraperitoneal, andcombinations thereof.
 8. The method of claim 6 wherein the N.meningitidis capsular polysaccharide is derived from at least one N.meningitidis serogroup selected from the group consisting of serogroupsA, B, C, D, X, Y, Z, 29E, W-135, and combinations thereof.
 9. The methodof claim 8 wherein the N. meningitidis polysaccharide is derived from N.meningitidis serogroup C.
 10. The method of claim 6 wherein thepneumococcal protein is selected from the group consisting of of PsaA,pneumolysin, PspA, PspC, CbpA, pneumococcal histidine triad proteinselected from the group consisting of PhtA, BVH11-3, PhtB, PhpA, BVH-11,PhtE, BVH-3, PhtD and BVH-11-2, PcpA, PppA, Dpr, NanA, NanB, PiuA, PiaA,LytA, LytB, LytC, ClpP, PavA, and combinations thereof.
 11. The methodof claim 10 wherein the pneumococcal protein is PsaA.